Sex differences in the cellular differentiation of the developing brain when paired with altered development can lead to abnormal circuitries that may be associated with sex differences in susceptibility to major mental disorders. The goal of our studies is to determine where and how genetic and hormonal factors interact to result in sexual differentiation of the brain. This proposal concentrates on the formation of the preoptic area/ anterior hypothalamus (POA/AH) as a key target for studying this process. The POA/AH is important for regulating homeostatic, neuroendocrine, and behavioral functions. It is a region where hormones dramatically influence development and where hormone-concentrating cells regulate physiology and behavior. We hypothesize that gonadal steroid hormones may "overwrite" direct genetic instructions, and a hormone-free development allows evaluation of the underlying genetic instructions. Mice with a disrupted steroidogenic factor-1 (SF-1) gene never develop a gonad and provide a model to test the hormone- dependence versus independence of selected characteristics of brain sexual differentiation. Our methods render developmental processes accessible to live observation, and direct manipulation in vitro. We can raise SF-1 knockout (KO) mice to adulthood to assess adult consequences of brain sexual differentiation without endogenous gonadal steroids. Three major questions will be addressed: 1) How are sex differences in the positioning of cells in the embryonic POA/AH regulated by gonadal steroids at different ages? 2) Does nitric oxide generation contribute to the development of the POA/AH by influencing the characteristics of cell migration, cell death, or the phenotypic differentiation of cells? 3) What are the functional consequences of developing in the absence of exposure to gonadal steroids? These questions will be addressed using unique transgenic and gene disrupted models that mark specific cell populations for fluorescence and prevent endogenous gonadal steroid hormone synthesis or prevent neural generation of nitric oxide. The proposed studies will bring us closer to determining the hormone-dependent and independent steps involved in the establishment of sex differences in brain structure and function.